The purpose of this project is to understand the molecular mechanisms involved in the replication of picornaviruses in susceptible target cells. This virus family includes numerous human pathogens (poliovirus, coxsackievirus, echovirus, rhinoviruses, hepatitis A virus). Infection of cells with these viruses leads to drastic changes in the host cell's structure and metabolic activity: Cellular protein and RNA synthesis are inhibited;the intracellular membrane network becomes rearranged into a network of vesicles that surround and provide a scaffold for viral RNA replication complexes;cellular proteins are subverted into facilitating viral protein and RNA synthesis. The unique combination of viral and cellular proteins together accomplishes a highly efficient production of viral RNA, proteins, and particles. We previously developed a method to select for sites in picornaviral proteins that are able to tolerate the insertion of extra amino acids while still retaining viability of the viruses that carry these insertions. During FY2009, we identified sites in four viral proteins essential for viral RNA replication and have begun screening for sites in two additional proteins. We have inserted fluorescent protein tags into one of these replication proteins, characterized the resulting viruses and performed intracellular localization and live cell imaging to observe the dynamics of protein movement during infection. In another viral protein, we inserted an epitope sequence that allowed us to identify both viral and new cellular protein binding partners that participate in viral RNA replication. Another key viral replication protein was purified and its biochemical activities and biophysical properties have been analyzed. We have shown that this protein forms oligomers of 6-8 subunits, and that the oligomeric protein is required for ATPase activity. We have identified the biochemical determinants for oligomerization and constructed mutant protein forms that are unable to oligomerize in order to study the loss of function(s) of this protein. Finally, we have made great progress in understanding the structure and function(s) of the membrane-associated replication complexes that are induced to form in infected cells. Viral proteins that trigger different aspects of membrane remodeling have been identified, and a cellular protein, guanine nucleotide exchange factor (GBF1) has been shown to participate in the RNA synthesis function of the newly-formed replication complex. This latter finding represents a new cellular pathway in which a host cell factor has been identified on which poliovirus (and other positive-strand RNA viruses) replication depends. Three-dimensional tomographic electron microscopy is in progress to reconstruct the three dimensional stucture of the replication complex membranes.